Cancer Research Techniques

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Protein Tyrosine Kinase and Phosphatase Expression Profiling in Cancer Research

Tyrosine kinases (PTK) represent only 10% of all protein kinases, although they are the most important protein kinases. PTKs are involved in cell growth signaling. Protein tyrosine phosphatases (PTP) attenuate growth signals generated by the PTKs through catalyzing the tyrosine dephosphorylation step on their substrate proteins. PTPs also are fundamental in cell cycle regulation and activation. Knowledge of the overall expression pattern of PTKs and PTPs in any given cancer cell represents the first step in understanding the sequential events of tumor progression.

Total RNA can be isolated from cells with any RNA extraction reagent (e.g. GenElute™ Mammalian Total RNA Miniprep Kit, RTN10), stored in 100% formamide (F9037) at -80 °C, precipitated with ethanol and redissolved in DEPC (D5758) treated water right before use. Immediately before PTK/PTP profiling, integrity and quality of purified total RNA should be assessed by gel electrophoresis or by PCR with positive-control actin gene primers.

Incubate at 42 °C in a water bath for 30 min. Stop the reaction by adding 1 µL 0.5 M EDTA solution.

Bring the total volume to 65 µL by adding DEPC H2O, and remove RT enzymes by phenol/chloroform (P1944) extraction once and chloroform (C7559) extraction once. Transfer the aqueous phase to a fresh Eppendorf tube.

Spin the CHROMA spin-200 column to remove buffer, and load the first strand RT reaction mixture onto the CHROMA spin-200 column. Centrifuge the column again to separate the first strand cDNA products and unincorporated dNTP mixture and primers. Collect the effluent in a fresh Eppendorf tube, and store the first strand RT cDNA template at –80 °C if necessary.

PTK/PTP insert sequences of each plasmid are determined by DNA autosequencer. The sequences obtained can be compared with GenBank database sequences from National Center for Biotechnology Information using the BLAST program to identify each PTK/PTP gene expressed.

PTK/PTP expression profiles can be established by the number of different kinase/ phosphatase clones identified.

6.6 µL formamide loading dye is added to the digested products and incubated at 65 °C for 7 min. before loading onto gel.

4 µL of final product is applied to each lane in 0.7% denaturing sequencing polyacrylamide gel. A sequencing reaction product (35S-label, and single-track sequencing only) with a predetermined sequence template is used as a standard for fragment size throughout the profile analysis. Any known sequence will be useful, because we use it as a size standard. (see Notes 3-5.)

Following electrophoresis, the gel is dried and exposed to a X-ray film or processed by a phosphoimager.

The amplified PTK products are digested with Mwo I restriction enzyme and separated on a sequencing gel. Samples from multiple tissues could be displayed on a single sequencing gel; thus, we could effectively screen all known human PTKs/PTPs in a short period of time.

From the pre-established bioinformatics database of human PTKs/PTPs digested with different restriction enzymes, individual tyrosine kinases were identified based on their respective characteristic restriction fragment sizes on the exposed X-ray films or in the phosphoimager-processed files.

PTK/PTP expression profiles can be established by the radiation dose unit numbers of different kinase/phosphatase identified. (see Note 6.)

The catalytic domains of PTK and PTP can be divided into a number of smaller sub-domains that represent localized regions of high conservation (3,4). By using degenerate primers from these regions, as well as the RT-PCR strategy, we could easily amplify most PTK/PTP genes expressed in cells, if not all PTKs/PTPs. The first PTK/PTP profiling method (see Methods 2) required cloning and sequencing hundreds of clones following the RT-PCR reactions (5, 6). Although it was an effective approach, it was time consuming for sequencing hundreds of clones. In addition, this protocol could not generate a more comprehensive and quantitative PTK/PTP profile until thousands of clones were sequenced. However, it is suitable for screening novel PTK/PTP genes in organisms without comprehensive genome sequence information as previously demonstrated by others (7,8,9).

In this second PTK/PTP display approach (see Methods 5), the expression level of PTK kinase genes could be represented by the intensity of each recognized band. This protocol is established on the bioinformatic restriction digest databases, which require the sequences of almost all PTKs/PTPs. Otherwise, there will be many unknown fragments on the gel and thus severely diminishes the effectiveness of this display approach. Although one can excise the fragments of interest from gels and identify them by cloning methods (10), it is time consuming and less effective. Because it did not employ the previously used cloning and screening procedures, this display method can be quantitative and more representative by utilizing the radiolabeled primers (1,11).

Because the radioactive label is only on the forward primers, each radioactive signal is generated from one labeled primer. Another advantage of this method is that one can perform multiple PTK profiles on several cell lines or tissues at the same time, whereas it would be difficult to perform large amounts of screening with the previous cloning and sequencing method. As discussed before, a comprehensive database is essential for this display approach, which might be useful for human or mouse PTK/PTP profiling. One has to screen the genome sequence or EST databases to generate the restriction digest databases (12). We have now implemented bioinformatic programs to automatically screen the genome and generate restriction digest databases for display purposes. Another issue that could be resolved by bioinformatic tools is single nucleotide polymorphism (SNP) within the PTKs/PTPs.

Because the display approach depends on the restriction enzyme recognition, SNPs located in the amplified PTK/PTP regions will affect the restriction enzyme recognition and generate different digestion fragments. This is similar to restriction fragment length polymorphism. Generally, it is not an important issue for the PTK/PTP display approach, because we are using a panel of restriction enzymes to determine the identity of each PTK/PTP gene, not just one enzyme. In addition, similar size restriction fragments could represent several different kinases, and alternative restriction enzymes would be necessary to identify single gene specific restriction fragments. It was calculated that about 15-20 restriction enzymes (4-base or 5-base hitters) were required to cover all genes here. With the aid of bioinformatic software, we might be able to include SNP information in the databases in order to generate a better PTK/PTP profile. With completion of the human genome project, we could examine the entire human transcriptome and learn more about the composition and evolution of gene families in the human genome. This will greatly assist this display approach, because all restriction fragments or patterns could be assigned to every known gene.

Additional improvements can be achieved in primer design. We use the degenerate primers based on the conservative motifs of PTKs/PTPs to cover all known and unknown genes, and specificity is compromised with the degenerate primer design. Because we now have all the human genome sequences, new human profile primers can be designed to increase specificity and reduce cross-hybridization with other serine/threonine kinases or phosphatases.

One major drawback for this display protocol is the short half-life of radioactive isotope labeled primers. In the future, with the addition of fluorescent-labeled PCR primers, we can also include internal MW markers, control genes tagged with different color dyes in the same reaction. This will extend the usage of labeled primers, because there is no isotope half-life and re-labeling considerations. Quantitation of each band was easier and less time consuming with fluorescent-labeled primers. With 33P labels, exposure time to film or phosphoplate ranged from three to seven days. 32P can be used to reduce the process time (10), however, gel resolution is much reduced owing to the strong 32P radioactivity. Analysis could be performed immediately following gel separation with fluorescent labels on autosequencers.